The autoimmune inflammation of rheumatoid arthritis (RA) induces profound invasive and proliferative changes in the joint synovium. Our research has focused on the role of the cytokine MIF, which we initially cloned together with its two-component receptor and found to sustain inflammatory responses by counter-regulating glucocorticoid action and inhibiting activation-induced apoptosis. We identified functional polymorphisms in the MIF promoter that are associated with disease severity and we developed an anti-MIF monoclonal antibody that has entered phase I clinical testing. While investigating the mechanistic basis for phenotypic differences between MIF and MIF receptor deficient mice, we discovered a new MIF-like cytokine encoded by the Ddt locus. Our data indicate that DDT is expressed in RA and exerts immunoregulatory functions by interacting with the CD74, ligand- binding component of the MIF receptor. DDT shares structural features with MIF but there are differences that are important in considering the therapeutic inhibition of these cytokines or thei signaling pathways. In this renewal application, we will determine the distinct immunologic actions of the novel cytokine DDT by pursuing three Specific Aims: 1. Define the Mechanism of DDT Signal Transduction. We hypothesize that DDT initiates signal transduction via the canonical MIF ligand-binding receptor CD74, and that it recruits additional co-receptors (CD44, CXCR2/4) to mediate shared or unique effector responses. We will ask if DDT, which lacks the pseudo-(E)LR motif by which MIF interacts with CXCR2/4, activates these or related co-receptors. We also will examine if DDT shares MIF's role in suppressing insulin signaling, as metabolic dysfunction and atherogenesis are important sequela of long-standing rheumatoid inflammation. 2. Define the Functional Importance of DDT in Inflammatory Arthritis. We hypothesize that DDT exerts a non-redundant role in the immunopathogenesis of inflammatory arthritis. We will define the phenotype of DDT-KO and MIF-DDT double-knockout (DKO) mice in experimental arthritis and compare them to MIF-KO and MIF receptor-KO (CD74-KO) mice. We will examine cross-regulation of MIF and DDT signaling in monocytes and fibroblasts from corresponding genetically-defined mouse strains. 3. Evaluate the Therapeutic Potential of Pharmacologic DDT Inhibition. We hypothesize that DDT neutralization ameliorates arthritis by innate and/or adaptive immune mechanisms. We will test a novel anti-DDT mAb and a soluble CD74-Fc fusion protein, which binds both MIF and DDT. We also will test the therapeutic activity of a small molecule, selective DDT inhibitor identified by computational screening. The discovery of DDT as a novel cytokine interacting with the MIF receptor CD74 was unforeseen but requires close examination for a better understanding of these cytokines in synovial inflammation and for the effective application of pharmacologic inhibitors.